The emergence of human mesenchymal stem cell (hMSC)-based therapies requires the growth of a large number of viable, clinical grade hMSC for transplantation and their subsequent delivery into a specific site. Current methods for both large-scale growth on the surface of tissue culture plastic and for subsequent cell harvest using trypsin are both inefficient for cell expansion and transplantation and present opportunities for contamination during passaging. Historically, microcarriers have been used with other cell types as three- dimensional substrates in a bioreactor to provide efficiency in space utilization as well as ease in assessing cell culture status. While microcarriers are currently available commercially, they are suboptimal for hMSC expansion since they do not have the specific attachment signals that the hMSC hyaluronic-acid rich, extracellular matrix typically provides. Additionally, some microcarriers are animal collagen-based, causing regulatory concerns of animal-transmitted disease in subsequent cellular delivery. The ideal cultivation system would allow growth of hMSC requiring less space and limiting the potential for contamination, would be physiologically relevant, and would facilitate subsequent hMSC transplantation. Glycosan BioSystems has assembled a unique team of biomaterial and hMSC cultivation experts that propose to use HyStem hydrogels, a chemically modified and crosslinked form of hyaluronan, to produce animal-free microcarrier hydrogels for the expansion and seamless transplantation of hMSC. These microcarriers would not only provide the requisite attachment sites for hMSC and thereby increase expansion efficiency but would serve as a delivery vehicle for cellular therapies. HyStem hydrogel have been shown to increase implanted cell engraftment and survivability [23]. The assembled team includes Dr. Glenn Prestwich, the director of the University of Utah's Center for Therapeutic Biomaterials and world recognized expert in glycosaminoglycans research, Dr. Linda Kelley, the director of the University of Utah Cell Therapy Facility and Professor of Medicine and Hematology, and Dr. Sarah Atzet, Director of Research and Development at Glycosan BioSystems. This proposal is designed to demonstrate the feasibility of producing animal-free HyStem microcarriers and evaluating their efficacy with hMSC both in expansion and cell delivery. In our first and second aims, we will synthesize recombinant thiol-modified human gelatin (rhGelin-S) and optimize the matrix composition for performance in a stirred flask bioreactor. We will characterize the different synthesized microcarriers physically and biologically. In our third aim, we will assess the in vivo performance of hMSC laden microcarriers in an osteochondral defect murine model. Phase II research will focus on scaling up both the production of hMSCs on microcarriers in industrial scale bioreactors and of the microcarriers themselves in compliance with the cGMP requirements of the FDA. PUBLIC HEALTH RELEVANCE: The emergence of human mesenchymal stem cell (hMSC)-based therapies requires the growth of a large number of viable, clinical grade hMSC for transplantation and their subsequent delivery into a specific site. Current methods for both large-scale growth on the surface of tissue culture plastic and for subsequent cell harvest using trypsin are both inefficient for cell expansion and transplantation and offer opportunities for contamination during passaging. The goal of this proposal is to determine the feasibility of preparing biodegradable and biocompatible microcarriers that can double as a substrate for expanding hMSC in a bioreactor and as a delivery vehicle for localized applications.